PROJECT SUMMARY The new era of precision medicine to discover and act upon genetic findings has inadvertently created a huge clinical dilemma when a variant of uncertain significance (VUS) is encountered. Patients identified as BRCA or RAD51 VUS carriers undergoing genetic counseling experience confusion, anxiety, and the potential for misguided treatment plans. Current estimates indicate that between 10-20% of BRCA sequencing results are VUS representing thousands of patients in the U.S. alone. These numbers will likely escalate as precision medicine and commercially available genetic testing services are being deployed at a rapid pace. The BRCA2 and RAD51 proteins play central roles in the homology-directed repair (HDR) of DNA double-strand breaks (DSBs). Functional diagnostic tools to decipher HDR VUS are still lacking in the setting of medical oncology. This is a critical issue as germline HDR mutations predispose individuals to a high risk for cancer while somatic mutations identified in tumors can dramatically impact treatment plans. For example, synthetic lethal strategies employing PARP inhibitors are currently being exploited in oncology to selectively target HDR deficient tumors. Therefore, the correct interpretation of a somatic tumor VUS can guide therapy selection. Functional assays to determine the pathological outcome of VUS are urgently needed to provide clinical guidance regarding cancer risk and treatment options. Our long-term objective is to bring molecular and mechanistic clarity to the operative pathways that HDR utilizes to repair DNA breaks. By using a highly synergistic biochemical and genetic approach, we aim to elucidate how VUS impact BRCA2 and RAD51 functions. Our central hypothesis is that a cell-based and biochemical functional approach will successfully differentiate pathogenic from harmless HDR VUS. In aim 1, we will establish cell-based complementation assays from which clinically relevant agents can be used to identify pathogenic BRCA2 and RAD51 variants. Integrating tumor-derived somatic variants into our cell-based models will enable in vitro testing of second-line therapies or novel compounds informing clinical practice of patients who could derive benefit from targeted agents. In aim 2, we will determine how pathogenic variants alter the biochemical functions of BRCA2 and RAD51. Results will be used to further refine our mechanistic understanding of pathological HDR variants and how biochemical defects translate into increased cancer risk. Our approach is innovative because of our unique skill set and development of robust cell-based and biochemical functional assays to dissect HDR mechanisms focused on BRCA2 and RAD51. The proposed research is significant because it will lay the groundwork to provide patients and physicians with clinically actionable information regarding risk reduction steps and stratification for targeted therapies such as PARP inhibitors. Furthermore, studies of pathological variants will provide insight into the molecular pathways that go awry when HDR functions are compromised.